To improve 3-ketosteroid-△1-dehydrogenase (KSDH) activity and the transformation level for androst-4-ene-3,17-dione,3-ketosteroid-△1 -dehydrogenase gene (ksdD) from Arthrobacter simplex was cloned into plasmid pWB980 and expressed in B. subtilis WB600 under the control of promoter P43. The molecular weight of expressed enzyme was about 55kDa by SDS-PAGE analysis. The activitities assayed by spectrophotometrical method of intracellular and extracellular soluble enzyme were 110 ± 0.5mU and 15 ± 0.6mU per milligram of protein respectively. The transformation rate of androst-4-ene-3,17-dione by the B. subtilis recombinant cells was 45.3%. Compared with Arthrobacter simplex, the enzyme activity of KSDH expressed in B. subtilis was improved about 30 fold, and the transformation level of androst-4-ene-3,17-dione by the B.subtilis recombinant cells was improved about 10 fold. The recombinant B. subtilis cells used in biotransformation of steroids provided a new way for steroid medicines production.

Human papillomavirus continuous (HPV) infection is an essential factor to induce cervical intraepithelial neoplasia and cervical cancer.Treating HPV infection is considered as a starting point to develop an effective therapeutic vaccine, which is a new strategy for prevention and treatment of cervical cancer.In recent years, development and trials of therapeutic HPV vaccine have made great progress.Selection of vectors, utilization of adjuvants, synthesis of fusion proteins and chimeric proteins have been widely applied to research to enhance vaccine immunogenicity, to increase vaccination safety, to reduce the side effect and so on.The clinical trial results are encouraging: various types of vaccines can induce a specific immune response with good tolerance.However, numerous studies are still required to obtain further success.In addition, HPV exists in various forms, thus it is also the focus of study to expand the range of action and reduce immune escape.

Objective To determine the persistence time of genotype 4 hepatitis E (HE) viremia after the onset of clinical symptoms in HE patients and provide essential data for study on HE epidemiologieal transmission, so that to evaluate potential contagiousness of HE patients after clinical stage. Methods The first serum samples from 162 HE patients after hospitalized in Eastern China were collected and tested for hepatitis E virus (HEV) RNA by nested reversed transcription- polymerase chain reaction (RT-PCR). The persistence time of HEV viremia after the onset of clinical symptoms was estimated with Kaplan-Meier survival analysis. Results HEV RNA was detectable in 101 out of 162 serum samples with positive rate of 62.35%, which was all grouped to genotype 4 by homology analysis. Furthermore, HEV RNA was detectable in 74 (64.91%) out of 114 male and 27 (56.25%) out of 48 female, which was not significantly different (χ2 = 1.08, P=0. 30). Kaplan-Meier survival analysis showed that the median persistence time of HEV genotype 4 viremia was 24 days after the onset of clinical symptoms (95% CI: 18-30 days), which meant that the viremia of 50% HE patients remaining detectable up to 24 days after the onset. The 75% and 25% percentiles were 14 days and 31 days, respectively. There was no significant difference of viremia persistence time between male and female (Breslow test: P=0.98, Tarone-Ware test: P=0.91). Conclusions The viremia of 75% patients with HEV genotype 4 infection could persistent until 2 weeks after the onset of clinical symptoms and that of some patients could persistent over 1 month. It is indicated that the viremia is still persistent and HE patient could be a reservoir even after the clinical symptoms disappeared and biochemical marks normalized.

Acute basilar artery occlusion (ABAO) has a high rate of disability and mortality, and the key to its treatment is to start reperfusion therapy as early as possible. A number of retrospective studies have found that the good prognosis rate of endovascular treatment of ABAO is related to number of infarction locus, extent of ABAO, vascular occlusion and collateral circulation. Screening patients through imaging evaluation before endovascular treatment may further improve the rate of favorable outcome. This article reviews research progress on the correlation between imaging evaluation before endovascular treatment of ABAO and clinical prognoses.

Objective:To explore the effect of dental specialist medicine and community linkage model on oral healthcare cognition and self-management level of residents.Methods:By the convenient sampling method, a total of 100 long-lived community residents in 5 communities within the jurisdiction of Changsha Stomatological Hospital from January 2015 to December 2018 as the research objects. According to the random number table method, they were divided into the control group and the observation group, with 50 cases in each group. The control group was given health education of dental specialist medicine while the observation group was given community linkage intervention based on the control group. The levels of oral healthcare cognition and self-management and satisfaction were compared between the two groups.Results:After intervention, the oral healthcare cognition score of residents in the observation group was (86.42±24.23) , higher than that (75.79±22.51) of the control group, and the difference between the two groups was statistically significant ( P<0.05) . After intervention, the score of Self-Management Scale in the observation group was (156.45±23.76) , which was higher than (124.59±20.42) in the control group, and the difference between the two groups was statistically significant ( P<0.05) . The satisfaction of residents with the intervention mode in the observation group was 98.00% (49/50) , higher than that in the control group 84.00% (42/50) , and the difference between the two groups was statistically significant ( P<0.05) . Conclusions:Dental specialist medicine and community linkage model both has positive effects on improving oral healthcare cognition and self-management level of residents.

Objective To investigate the inhibitory effect of a disintegrin and metalloprotease 12 silenced by shR?NA on self-renewal capacity of CD133 positive giloma cells. Methods The shRNA recombinant lentivirus aimed at si?lencing ADAM12 was prepared. Human glioma cells U87 were employed in this study and assigned into three groups:shRNA-ADAM12, shRNA-NCandshRNA-C. ADAM12 expression was detected at mRNA and protein level using Re?al-time quantitative-PCR and western bloting, respectively. U87 cells were cultured with stem cell culture medium, to obtain cell sphere formation in which CD133 positive glioma cells were enriched. Immunofluorescence was employed to detect the expression of ADAM12 and CD133 in cell spheres and U87 cells; Self-renewal was tested by using tumor sphere formation assay. Molecular markers for differentiated or undifferentiated cells (CD133,GFAP and Tuj1) were de?tected at protein using western blotting. Western blotting was employed to test protein expression of HES1. Results AD?AM12 shRNA significantly down-regulated the mRNA and protein expression levels of ADAM12. Compared with shRNA–C group, the relative expression levels of mRNA in shRNA-ADAM12 group and shRNA-NC group were 0.22 ± 0.03 and 0.98 ± 0.06 (F=425.37,P<0.01). The relative expression levels of protein in shRNA-ADAM12 group, shRNA-NC group and shRNA-C group were 28.72%±2.36%, 69.21%±3.92%and 69.04%±3.57%, respectively (F=145.42,P<0.01). Immunofluorescence staining showed that expression levels of ADAM12 and CD133 in cell spheres were significantly higher than those in normal cells. The number of spheres in three groups were 45.5±2.3、104.2±5.8 and 109.6±6.2, tumor sphere formation ability of shRNA-ADAM12 group was lower than that of shRNA-NC group and shRNA-C group (F=147.03,P<0.01). Compared with the shRNA-NC group and shRNA-C group, the protain expression of GFAP and Tuj1 were increased up to 166% and 146% (P<0.01) whereas the protein expression levels of CD133 and HES1 were down-regulated by 54% and 50% (P<0.01). Conclusion Knockdown of ADAM12 may suppress self-renewal ability of CD133 positive glioma cells by inhibiting the Notch pathway activity.

Exosomes, as a type of multivesicular bodies secreted by various cells in human body, exist in many kinds of body fluids in the body. Exosomes have the structure of lipid bilayer membranes to protect the structure and function stability of their contents and they carry a large number of active substances including microRNA (miRNA) and mRNA. Studies have shown that exosomes and their miRNA are closely associated with the development and progression of liver fibrosis and regulate such process by acting on hepatic stellate cells to change their activation, proliferation, apoptosis, and migration. This article elaborates on the biological characteristics of exosome miRNA and its association with liver fibrosis, analyzes its important role in the development and progression, diagnosis and treatment, and prognostic evaluation of liver fibrosis, and points out that exosome miRNA may become a new potential target for the diagnosis, treatment, and prognostic evaluation of liver fibrosis.

Objective:To investigate the effects of fluoride exposure on proliferation, apoptosis and osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) in mice.Methods:BMSCs were isolated and cultured from femur bone marrow of C57BL/6 mice (6 - 8 weeks). The cells in passage 3 were used to detect the surface markers of stem cells by flow cytometry. The cells were cultured in media with a final fluoride concentration of 0.0, 0.1, 1.0, 5.0, 10.0, 15.0, 20.0 and 40.0 mg/L, respectively. The effects of different fluoride concentrations on BMSCs cell proliferation (CCK8 method), apoptosis (flow cytometry analysis), osteogenic differentiation ability [alizarin red and alkaline phosphatase (ALP) staining] were detected. Western blot was applied to detect the levels of apoptosis-related proteins [poly ADP-ribose polymerase (PARP)], mitogen-activated protein kinase (MAPK) pathway member proteins [extracellular regulated protein kinase 1/2 (ERK1/2), c-Jun amino-terminal kinase (JNK), p38 and phosphorylated ERK, JNK, p38 (p-ERK, p-JNK, p-p38)], osteogenic differentiation-related protein [Runt-related transcription factor 2 (Runx2), ALP] and Wnt/β-catenin pathway member proteins [glycogen synthase kinase-3β (GSK3β), phosphorylated GSK3β (p-GSK3β) and β-catenin]. Immunocytofluorescense staining was applied to evaluate the expression levels of p-GSK3β and β-catenin. The two pathways (MAPK and Wnt/β-catenin) were blocked by SP600125 and DKK-1, respectively, to testify their involvement in mechanisms of apoptosis and osteogenic differentiation.Results:The mouse BMSCs were successfully isolated and cultured. Flow cytometry analysis showed that the mesenchymal stem cell surface biomarkers (CD73, CD90 and CD105) were positively expressed. The comparison of cell proliferation at three time points (24, 48 and 72 h) in each concentration group was statistically significant ( F = 65.36, 160.04 and 365.32, P < 0.001), and the comparison of early apoptosis (24 h) in each concentration group was statistically significant ( F = 214.04, P < 0.001); compared with the 0.0 mg/L group, the cell proliferation in 15.0, 20.0 and 40.0 mg/L groups decreased, and the early apoptosis rate in 10.0, 15.0 and 20.0 mg/L groups increased ( P < 0.05). When cells were treated with 15.0 mg/L fluoride for 0 - 24 h, the p-JNK/JNK ratio was higher at 2, 4, 8, 12, 18 and 24 h compared with that at 0 min ( P < 0.05); compared with the fluoride group (15.0 mg/L), the early apoptosis rate of cells after SP600125 block decreased ( P < 0.05), and the protein expression levels of PARP and p-JNK decreased ( P < 0.05). After osteogenic induction, compared with the 0.0 mg/L group, in 0.1 and 1.0 mg/L groups ALP staining was enhanced and the number of calcified nodules increased, and the protein expression levels of Runx2 and ALP in the 0.1 and 1.0 mg/L groups were higher ( P < 0.05). After osteogenic induction, compared with the 0.0 mg/L group, the p-GSK3β/GSK3β ratio and β-catenin protein level were significantly higher in the 0.1 and 1.0 mg/L groups ( P < 0.05); and compared with the fluoride group (1.0 mg/L), addition of DKK-1 significantly decreased the protein expression levels of p-GSK3β and β-catenin and reduced the nuclear entry of β-catenin, and ALP staining decreased and the number of calcified nodules decreased. Conclusions:High concentration of fluoride (> 10.0 mg/L) inhibits the proliferation and promotes apoptosis of BMSCs, while low concentration of fluoride (0.1, 1.0 mg/L) promotes osteogenic differentiation. The MAPK/JNK pathway and the classical Wnt pathway are involved in the above cellular processes, respectively.

Objective:To investigate the effect and mechanism of PPAR-γ agonist Pioglitazone (PGZ) on the proliferation of malignant mesothelioma (MM) cells.Methods:In December 2019, MM cell lines MSTO-211H and NCI-H2452 were incubated with different final concentrations of PGZ (0, 10, 50, 100, 150, and 200 μmol/L) for different periods of time (24 h, 48 h, and 72 h) , and then the cell proliferation level was detected by CCK8 assay. After given various final concentration of PGZ (0, 10, 50, 100, 150, 200 μmol/L) the for 72 hours, the changes of number and morphology of MM cells were observed under an inverted microscope. The expressions of PPAR-γ and HMGB1 mRNA were determined by real-time fluorescence quantitative reverse transcription-polymerase chain reaction (qRT-PCR) after treatment of MM cells with PGZ of 0, 10, 50, 100 μmol/L for 72 h. The MM cells were treated with PGZ at concentration of 0, 100 μmol/L for 72 h, and the protein expressions of HMGB1 were examined using Western blotting and immunofluorescence; the protein expressions of Ki67 were assessed by immunohistochemistry.Results:The cell viability rate of MM cells was decreased after treated with PGZ ( P<0.05) . Cell number in PGZ-treated group was significantly less than that in control group and morphology changes were observed under light microscope. QRT-PCR results revealed significantly increased PPAR-γ mRNA expression in the PGZ-treated group compared to the control group ( P<0.05) . There was a significant decrease in the mRNA expression level of HMGB1 in the PGZ-treated group (100 μmol/L) as compared to the control group in MSTO-211H ( P<0.05) ; however, the expression level of HMGB1 in NCI-H2452 was an increase or no significant differences ( P>0.05) . Western blotting and immunofluorescence results showed that the protein expression of HMGB1 was reduced in the PGZ-treated group compared with the control group in MSTO-211H ( P<0.05) , but the protein expression of that in NCI-H2452 was no significant differences ( P>0.05) . Immunohistochemistry results showed increased expression of proliferation marker Ki-67. Conclusion:Pioglitazone suppresses the proliferation of MM cells through inhibition of HMGB1 by the activation of PPAR-γ.

Objectives To explore and compare the clinical characteristics and risk factors for mortality between patients with artificial quartz stone silicosis and those with classic silicosis. Methods A total of 48 patients with artificial quartz stone silicosis (experiment group) and 98 patients with classic silicosis (control group) were recruited as the research subjects using the convenience sampling method. Data of clinical symptoms, laboratory tests, high-resolution computed tomography (HRCT), and pulmonary pathology of the research subjects were retrospectively analyzed. The Cox proportional hazards regression model was used to analyze the influencing factors on the survival time of silicosis patients. Results Patients in the experiment group had shorter years of dust exposure, latency period and time since last exposure than those in the control group (all P<0.01). The positive rate of anti-nuclear antibodies and the expression of neuron-specific enolase in the experiment group were higher than those in the control group (39.6% vs 10.2%, median： 28.44 vs 16.25, both P<0.01). The PaO₂ levels in the experiment group were lower than those in the control group (median： 66.0 vs 89.0, P<0.01). The patients in the experiment group had lower vital capacity, inspiratory reserve volume, forced expiratory volume in the first second (FEV₁), forced vital capacity (FVC), and carbon monoxide diffusion capacity compared to the control group (all P<0.05), but the maximal expiratory flow in 75% vital capacity was higher than the control group (P<0.05). Compared with the control group, patients in the experiment group had the presence of ground-glass opacity (GGO) in both lungs, aggregation and fusion of subpleural nodules, and gradual formation of progressive massive fibrosis (PMF), with higher potential of pneumothorax. Within 5 years after diagnosis, the mortality of patients in the experiment group was higher than that in the control group (27.1% vs 4.1%, P<0.01). The Cox regression model analysis results showed that patients with nodule aggregation on lung HRCT images had a higher risk of mortality than those without nodule aggregation, and lower lung function including vital capacity, FVC, FEV₁ and maximum expiratory flow in 25% vital capacity had higher risk of reduced survival time (all P<0.05). Conclusion Compared with patients with classic silicosis, patients with artificial quartz stone silicosis have higher level of serum neuron-specific enolase, increasing the risk of autoimmune diseases. Pulmonary imaging features in patients with artificial quartz stone silicosis include GGO, PMF and susceptibility to pneumothorax, and rare calcification of mediastinal lymph nodes, leading to a higher mortality rate within 5 years after diagnosis.